For a start please record a fluorescence excitation spectrum (at 400 nm) and see whether it matches with the absorption spectrum of tryptophan.

I did emission @ 380 nm and excitation range from 200 to 350nm.  I got a spectra at 280nm and 330nm.  

You mean to do excitation my protein at 400nm? 

By "fluorescence excitation spectrum (at 400 nm)" I meant record a excitation spectrum monitoring the emission at 400 nm. I think you already did (at 380 nm)  what I suggested. The 280 nm signal is due to tryptophan, but what is the origin of 330 nm signal? Do you see the 330 signal in absorption  spectrum too? Peak normalize (at 280 nm) the absorption and excitation spectra and see where they are superimposable or not (pay special attention to 330 nm signal). Also compare the absorption spectrum with reported tryptophan absorption spectrum.

The higher emission spectra may be due to multimerization depends on the buffer you are using to some extent.  You do not say what concentrations or pH or the type of buffer you are running these spectra with.  Maybe try a lower concentration of tryptophan to see if there is variation in the ratio of emission peak heights.  It the higher emission peak falls as you reduce the tryptophan concentration then it is very likely to multimerization.  

Dear Prof.  Richard,  Thanks for your response.  The buffer I used was 30mM HEPES, 140mM NaCl, 40mM MgCl2 and 40mM KCl pH 7.2. The concentration of the protein was 10uM and the molecular weight of the protein was 38kDa with just 4 tryptophan.  As I mentioned earlier this was a truncated protein of a 61kDa protein.  The full length protein and other truncation doesn't give this pattern of spectra.  All the protein were done with same buffers only.

Dear Prof.  Richard,  you have recommended to lower the concentration of tryptophan.  I dont understand.  You mean to reduce the concentration of protein? 

Apologies I did not realise that you were dealing with tryptophan in a protein!  Ok, my answer still partially holds, the tryptophan emission could be altered by either inter or intramolecular hydrophobic interactions,   and can be tested by using denaturing agents e.g. high concentrations of either guanidinium hydrochloride or urea to see if this alters the emission spectrum and also reducing the protein concentration much as is compatible with obtaining a useable signal.   

Pi bonding interactions are commonly used in detecting conformational changes partial proteolysis could be a factor in producing a FRET signal and thus a change in the emission spectrum.  see attached 

Dear Prof. Richard,

Thanks for your prompt response. If I am not wrong, the emission of tryptophan won't go beyond 355nm during the red shift in denaturing conditions. But in this case there is a emission in 334 for tryptophan and the other peak is at 380nm.

Still I will check and update the emission under denaturing condition too to find the difference in the spectra soon.

From my further reading, the changes may have something to do with cation interaction with the aromatic residues, so maybe look at K Mg and Na concentration dependent changes too,

But I need these cations to do quenching studies, and moreover only in this truncation I have this issue.

Sure,  but it would be a good idea to determine if any of these cations are producing a signal with the truncated protein.  If they do have an effect on the signal,  then maybe you can quantify and compensate for changes.   

Thank you so much Prof. Richard, I will proceed as per your suggestion and update it. Thanks for sharing your experience and knowledge.

Dear Prof.  I have done the cation dependent changes as you suggest.  There is no any change in the spectra.  I have done emission at 380 and excitation between 200 to 350. I got 3 spectras at 236nm,  280nm and 310nm which says there is no problem other components in the buffer.  

That is interesting,  but don't you have to figure out why there is a difference in fluorescence emission between the fragment and the whole protein.  Are you saying that there was a difference in the buffer composition when you were measuring these samples.

Dear Prof.  The buffer compositions are constant to all the proteins.  Since I am getting two emissions in the truncation I checked with buffer compositions.  But nothing difference witnessed.  Is there any possibility of tryptophan tryptophan stacking,  tryptophan histidine stacking or something like this? 

Yes I think that is the most likely answer I have thought about this some more.  It might be possible to confirm this hypothesis by using high concentrations of urea  6M and or heating your solutions to 50-60C to see if unfolding makes a difference.  

Thanks for your suggestion Prof. Richard. I will do denaturation studies and will update the result in next few days..

Dear Angela, 

Thanks for writing,  I did denaturation of the proteins With 3M urea and 6M urea with the control of protein in buffer alone.  But still the peak with ~380 exist. Moreover the intensity of the peak got increased with the increased urea concentration. MAy I know how many histidine present in your protein and whether the side chain  presnt outside the surface of the protein or not. 

I tried with my blank alone too.  But there is no any peak found in the range of 300 to 500 nm. 

Kindly let me know if you found something intetesting... 

Dear Prof.  Richard,

As you suggested , i did denaturation before reading the spectra.  After denaturation the intensity of the peak is getting increased.. But still the second peak remains.  So I think our hypothesis was wrong.. 

The increase in absorption at 380 with urea sounds promising.  What was the percentage increase?   Sounds as if there might be quenching of the signal in the control sample.  

Dear prof,

I have not calculated the percentage.  I will let you know by tomorrow. 

If the signal in control is quenched, then  why we are getting a peak at 380nm and after denaturation the absorption at 335nm looks like shifting towards 380nm?

Dear Angela,

1. Yes!  My protein is an ATP binding protein which requires Mg for activation

2. I tried both with imidazole and without imidazole in my protein and blank.  But still i get two peaks..

Is your protein a metal binding protein? 

Hi everyone,

I am also experiencing a similar problem (with Antibodies). Did you find a conclusion for this? Very interested to see what you have found.

Thanks!

Jay

Your protein bind's ATP? Maybe it comes bound to your protein during purification. Take an absorption spectrum of your protein preparation. If ATP is bound, you're gonna see it as a shoulder around 260 nm, if i recall correctly. Moreover, Mg+2 binding (ATP stabilisation) should quenches this fluorescence quite a bit. Unloading ATP from your protein should fix this but in case where you want to mesure activity, it should be a problem; you must design your test properly. Also, you can check the fluorescence of free ATP to confirm suspicious contribution to emission spectra.

Good luck.